Cry毒素模拟物与昆虫潜在受体的分子互作规律研究

Since the use of Cry toxin genes in the breeding of insect resistant crops, the screening and exploration of novel insecticidal genes has been a hot research area in insect toxicology. The selection of anti-idiotype antibodies is a practical technique to create a mimic structure of an active center of a protein which has been verified in the development of many kinds of drugs in medicine industry. By combined using this technique with the phage display technology, we've got series of mimic materials of Cry toxins and showed different insecticidal activities. This project is designed to detect the different affinity and binding kinetics of the Cry toxin mimics with the putative receptors of Cry toxins. We are going to further generate massive amount of the improved mimics by combined use of directed evolution, site-directed mutagenesis, and molecular docking analysis. Then through high-throughput screening by using the connected “anti-idiotype antibody-receptor" system, combined with the molecular interaction analysis, we want to find out the specific antigenic epitopes of the binding regions or single amino acid in the active regions of the mimics of Cry toxin domainⅡ and Ⅲ firstly. Then we'll try to carry out the receptor-oriented screening of different kinds of the expression products by the gene splicing of the domain I, to reveal their binding affinity and its molecular interaction with the receptors. Finally, we hope to finish the preparation of high efficient candidate pesticides, by circular bioassays on several key insect pests, to characterize the key active structures of the analogues and its molecular interaction with the putative receptors, which may provide reference for the research and development of alternative materials of the Cry toxins.

自从利用Cry毒素基因转基因培育抗虫作物品种以来，各国科学家围绕新杀虫材料的筛选与创制进行了广泛探索。多种药物研制已经证明：利用抗独特型抗体技术模拟制备关键活性结构是一项实用的生物模拟技术。前期我们利用该技术已成功从抗体库中制备了具杀虫活性的Cry毒素模拟材料。本项目拟围绕进一步改进模拟物的杀虫活性，运用定向库进化，定点氨基酸替换与分子对接技术，定向制备毒素模拟物。创建高通量“抗独特型抗体-受体”组合筛选体系。对获得的高亲和力模拟物做分子互作分析,首先探明结构域Ⅱ和Ⅲ模拟物的关键结合区特征表位与氨基酸组成，再通过结构域I的基因拼接表达，借助靶向筛选，揭示其结构与受体结合的亲和力与分子互作特性。经多轮害虫杀虫活性测定，制备高杀虫活性模拟物。在此基础上，总结高杀虫活性模拟物的关键活性结构特征，及其与潜在受体的分子互作规律，为研发人源化安全Cry毒素模拟杀虫材料提供理论依据。

项目组完成了Cry1Ab、Cry1Ac、Cry2Aa 、Cry1B、Cry1F和Cry1C等6种毒素模拟物对象的材料制备，分别从“关键结合位点氨基酸-毒素关键结合区-毒素不同结构域-毒素整体结构”等4个层次（类型）模拟物材料库制备。其间，借鉴了循环定向设计，通过构建中间材料进化库，或借助对接工具进行了分子设计优化，完成了模拟物材料库的活性定向筛选，80余个重点模拟物材料进入活性互作研究。完成了Cry毒素对昆虫中肠BBMV和当前主要潜在受体：钙粘蛋白CAD、碱性磷酸酯酶ALP、氨基肽酶APN、ABCC2和一种新的脂质运载蛋白、V-ATPaseA亚基等的材料制备，开展了原核、真核及果蝇品系构建工作，并对上述制备材料的受体结合活性、细胞毒性与杀虫活性进行了测定，完成对小菜蛾、棉铃虫、稻纵卷叶螟、二化螟、玉米螟和草地贪夜蛾等重要农业害虫的潜在毒素受体结合与个体生物活性评价及关联分析。完成高受体结合活性材料的分子互作特征分析与分子互作能量分析。初步揭示了不同类型Cry毒素模拟物材料的结构与杀虫活性的关系，并获得了3种类型12种具有应用潜力的Cry毒素模拟物材料。项目工作也验证了抗原表位模拟技术在毒素模拟物制备应用的可行性，提出了不同模拟物关键结合结构与不同受体结合靶点对模拟物杀虫活性的多靶点互作效应。为更进一步深入认识毒素模拟物与受体的分子互作机制及创制更加安全高效的杀虫活性模拟物材料工作提供多方面阶段性成果。